Phenol-Chloroform Extraction (DNA) - (Oct/31/2013 )

Hi again,

I am looking for a protocol for phenol-chloroform extraction of DNA from tissue (rat tail). I found a lot of protocols online but none of them specifies the extraction procedure. They more likely give the purification steps. 

I want to know how do i treat the tissue for DNA extraction.

Looking forward

Cheers,

here are several: http://www.protocol-online.org/prot/Genetics___Genomics/Transgenic_Techniques/Transgenic_Mice/Transgenic_Identification/Mouse_Tail_DNA_Extraction/index.html

Thanks mate.

I had a very quick question. Can i substitute ethanol for Isopropanol (we ran out of isopropanol).

Cheers

If this is the DNA precipitation stage, then yes, it can be substituted. For isopropanol, you need only 0.6 volumes for precipitation. For ethanol, you need to add 2.5 volumes, so you need a larger tube to spin the precipitate in (or you can divide it).

  I am talking about the 7th step in this protocol

Cheers,

Obtain the last 1 to 3 mm of the mouse tail and place directly into an eppendorf. 

2. Add 200 μL of Digestion buffer. 

3. Add 2 μL of Proteinase K (10 mg/mL) <> 

4. Stir at 800 rpm for +/- 6 hours or 600 rpm overnight, both at 55 ºC. 

5. Centrifuge at 14000 rpm for 15 min. 

6. Remove supernatant (≈ 180 μL) and put it in a new eppendorf. Note: while you remove it, avoid bring together mouse piles. 

7. Add 180 μL of isopropanol (volume 0,7). Note: isopropanol is easily evaporated, so do not let flask open for big time. 

Yes, instead of adding 0.7 volumes of isopropanol, you  can add 2.5 volumes of ethanol.